[Biosynthesis of bacterial cellulose from glucose selectively deuterated in position 6: NMR study]. / Biosynthèse de cellulose bacterienne à partir de glucose sélectivement deuterié en position 6: étude par RMN

D-glucose specifically deuterated at C-6 was prepared and used for the biosynthesis of bacterial cellulose with Acetobacter xylinum. The material obtained was converted into glucitol hexaacetate and analyzed by 250 MHz n.m.r. and mass spectrometry. These spectra indicated that about 70% of the starting D-glucose was incorporated without modification of deuteriation at the C-6 position. However an explanation is required of the finding that deuterium was also incorporated at the C-2 and C-1 positions of the cellulose, arising respectively from the 6 pro-R and 6 pro-S positions of the initial D-glucose.

One-bond carbon-proton coupling constants: angular dependence in alpha-linked oligosaccharides.

An investigation is presented of the angular dependence of 1JC,H in model compounds related to alpha-linked oligosaccharides. Values calculated by FPT formulation in the semi-empirical INDO method for models of (1----1)-, (1----2)-, (1----3)-, and (1----4)-linked disaccharides were compared, and the effect of the orientation of HO-2 was elucidated. The angular dependence of 1JC,H on the glycosidic dihedral angles phi H and psi H was characterised in the form 1JC,H = A cos 2 phi + B cos phi + C sin 2 phi + D sin phi + E. The 1JC,H values, measured by DEPT methods for C-1-H-1 and C-X'-H-X' in various rigid carbohydrates, were used to adjust the calculated angular dependences and to determine solvent effects. Based on the abundance of the conformers for maltose and isomaltose, the thermodynamically averaged 1JC,H values have been calculated. The results obtained (less than 1JC-1,H-1 greater than 169.9, less than 1JC-4',H-4' greater than 147.7 Hz for methyl beta-maltoside and less than 1JC-1,H-1 greater than 169.8 Hz for methyl beta-isomaltoside) agree with the experimental values of 172.4, 147.7, and 170.3 Hz, respectively.

Conformational study of digalacturonic acid and sodium digalacturonate in solution.

The solution conformation of digalacturonic acid and its sodium salt have been analyzed using nuclear magnetic resonance data and molecular mechanics calculations. The flexibility around the glycosidic linkage was characterized by calculation of the relaxed (phi, psi) potential surfaces for the isolated molecule, and also for dimethyl sulfoxide and aqueous solutions using the CHARMM and SOLVOL programs. The one-bond and three-bond proton-carbon couplings were measured and H-1'-H-4 distances were estimated from NOESY experiments. The calculated potential surfaces were used to determine theoretical ensemble averages of NMR data. The agreement between the experimental and theoretical data is very satisfactory. The calculations show a strong effect of solvent on the solution behavior of both compounds. The vacuum lowest energy conformer of digalacturonic acid is stabilized by solvation, while for sodium digalacturonate the solvent induces a conformational change. An extrapolation of the stable conformers to polysaccharide chains implies that poly(galacturonic acid) occurs in solution as a three-fold helix and sodium poly(galacturonate) as a two-fold helix.

Cassia spectabilis DC seed galactomannan: structural, crystallographical and rheological studies.

The seeds of Cassia spectabilis DC (family: Leguminoseae), an Indian fast growing spreading tree, contain about 40% of endosperm and possess the characteristics of becoming a potential source of commercial gum. The purified galactomannan shows Mw 1.1 x 10(6), intrinsic viscosity [eta] 615ml/g with k' = 1.706 x 10(-1), and a mannose to galactose ration of 2.65. The hydrolysis of the fully methylated polysaccharide reveals clearly the expected structure of legume galactomannans. The orthorhombic lattice constants of the hydrated gums are as follows: a = 9.12 A, b = 25.63 A and c = 10.28 A. The results of X-ray fiber studies show that the b dimension of the unit cell is very sensitive to relative humidity (RH), galactose substitution and orientation of the films. The probable space group symmetry of the unit cell is P2(1)2(1)2. Rheological studies of the galactomannan have shown that the transition from semi-dilute to dilute regime occurs at a critical concentration Cc* = 2.75. The slope of the log-log plot of specific viscosity versus C at zero shear rate is 5.87 in the more concentrated regime. The viscoelastic and critical shear rate behavior indicate the characteristics of a coil polymer. The large dependence of the viscosity on the coil overlap parameter is probably due to polymer-polymer interactions and peculiarity of the galactose distribution along the chain. Above 20 g/L concentration, the rheological behavior of the gum is like the one of a weak-gel.

Conformational analysis of carbohydrates inferred from NMR spectroscopy and molecular modeling. Applications to the behavior of oligo-galactomannan chains.

In both the biological functions and industrial applications of carbohydrates, their conformational behavior along with the dynamic fluctuations that their structures experience are of great significance. The elucidation of the three-dimensional characteristics of carbohydrates and carbohydrate-containing molecules is in general performed by a combination of high-resolution nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. Heteronuclear couplings and nuclear Overhauser effect (NOE) data are major tools for structural determination. The angular dependence of carbon-proton coupling constants has been shown to depend also on the pathways involved. Concerning molecular modeling, the goal is to generate the ensemble of structures that are consistent with the experimental data. This approach is illustrated in the galactomannan chain. It consists of the conformational study of two model disaccharides (mannobiose and epimelibiose) of galactomannans through carbon-proton coupling constants and NOE measurements. Experimental data are then compared to results of conformational analysis that predicted the stable conformers in solution. From these results helical conformations of oligomers are generated and are discussed in terms of the influence of the position and number of galactose units on the conformation of the mannan backbone.

N.m.r. studies of synergistic kappa carrageenan-carob galactomannan gels.

C-n.m.r. spectroscopy has been used to investigate the carob galactomannan-kappa carrageenan binary gels. Starting from partially depolymerized carob samples, evidence for interaction and intermolecular binding was found by analysis of the spectra obtained in quantitative conditions and in the absolute intensity mode. From these, a reconstitution of the signals corresponding to C-1, C-4, C-5 and C-6 showed the existence of three kinds of galactomannan chains; the first ones with a low galactose content were strongly connected and as such had completely lost their mobility; but the chains with a high galactose content were still detected and could be divided in two groups according to their mobility.

Synergy of the kappa-carrageenan-carob galactomannan blend inferred from rheological studies.

Rheological measurements have been used to investigate kappa-carrageenan-carob galactomannan mixed gels at different ratios, in particular for very low kappa-carrageenan concentrations. The viscoelastic properties of the gels were explored using sinusoidal tests on dynamic rheometers and compression tests between parallel plates. The properties of the binary gels were compared with the kappa-carrageenan gels and with the carob galactomannan solutions. These measurements display significant synergy for very low kappa-carrageenan concentrations. From these data, it appears that the synergy is based on a gel structure defined by a coupled network with specific junction zones. However, above a total polysaccharide concentration of 8-10 g l-1, self-association of galactomannan chains could also take place.

Conformation and dynamics of a cyclic (1-->2)-beta-D-glucan.

A molecular modelling and nuclear magnetic resonance spectroscopic study was performed in order to gain insight into the conformational preferences of cyclosophoroheptadecaose. MM3 molecular mechanics calculations predicted a non-symmetric conformer with a small cavity of 3.7 A diameter as the lowest energy form. Molecular dynamics simulations gave insight into the dynamics of the free cyclosophoroheptadecaose and also supported the results of molecular mechanics calculations. A fair agreement was found between experimental data and corresponding average values predicted by molecular modelling.

Single crystals of inulin.

Lamellar crystals of inulin were grown by crystallizing sharp fractions of low molecular weight inulin from dilute aqueous ethanol solutions. The crystals were analyzed using three-dimensional electron diffraction and X-ray powder diagrams. Two crystalline polymorphs were observed, depending on the hydration conditions: a hydrated form which indexed on an orthorhombic unit cell, with space group P2(1)2(1)2(1) and with cell dimensions of a = 1.670 nm, b = 0.980 nm and c (chain axis) = 1.47 nm, together with a pseudo-hexagonal semi-hydrated form with unit cell parameters a = 1.670 nm, b = 0.965 nm and c (chain axis) = 1.44 nm. These parameters, together with the density data, indicate that inulin crystallizes along a pseudo-hexagonal six-fold symmetry with an advance per monomer of 0.24 nm. The difference between the hydrated and the semi-hydrated unit cells does not seem to correspond to any change in the conformation of inulin, but rather to a variation in water content.

Characterization and function of pig intestinal sucrase-isomaltase and its separate subunits.

Papain-solubilized pig intestinal sucrase-isomaltase was purified to homogeneity in a four-step process with a yield of 50%. The substrate specificities of the two enzyme activities were studied together and separately after inactivation or inhibition of one of the activities. Michaelis constants, maximum velocities and time courses of hydrolysis of several substrates, in particular alpha-limit dextrins, were used to characterize this complex of alpha-glucosidases. The participation of the enzyme complex in the hydrolysis of alpha-limit dextrins and more generally in pathways for starch breakdown in the pig digestive tract is examined and discussed.

Computer modeling of polysaccharide-polysaccharide interactions: an approach to the kappa-carrageenan-mannan case.

A computer program SAINT has been developed for the investigation of the structure and for the prediction of minimum-energy structure of polysaccharide-polysaccharide complexes. The energy minimization is carried out on internal geometrical parameters--namely bond angles, torsional angles, and five parameters describing the mutual orientations of polysaccharide chains. For this purpose, the nonderivative method of conjugated directions is used. This procedure was applied to computer modeling of an idealized model of the binary gelling kappa-carrageenan and galactomannan system. It is shown that the interaction between two chains influences the structure of the individual polysaccharide molecule and that in the minimum-energy structures of the complex, the conformation of the chains does not correspond to the lowest energy.

(13)C nuclear magnetic relaxation study of segmental dynamics of the heteropolysaccharide pullulan in dilute solutions.

(13)C spin-lattice relaxation times (T(1)) and nuclear Overhauser enhancements (NOE) were measured as a function of temperature and magnetic field strength for the heteropolysaccharide pullulan in two solvents, water and dimethyl sulfoxide. The relaxation data of the endocyclic ring carbons were successfully interpreted in terms of chain segmental motions by using the bimodal time-correlation function of Dejean de la Batie, Laupretre, and Monnerie. On the basis of the calculated correlation times for segmental motion, the flexibilities of the pullulan chain at a repeat-unit level have been studied and compared with the segmental mobility of the homopolysaccharides amylose and dextran in the same solvents. The internal rotation of the free hydroxymethyl groups about the exocyclic C-5 [bond] C-6 bonds superimposed on segmental motion has been described as a diffusion process of restricted amplitude. The rate and amplitude of the internal rotation of the free hydroxymethyl groups were not affected by the local geometry of the pullulan chain.

Computer modelling of kappa carrageenan-mannan interactions.

Molecular modelling has been used as a theoretical approach to investigate the kappa carrageenan structure and its interactions with mannan chains. Calculations revealed the existence of six minima for the kappa carrageenan structure in solution. Two of them were very close to the structure found in the solid state. The methodology allowed the calculation of a theoretical counterpart of the structures based on x-ray fibre diffraction studies. In the second step of this study, we have shown that there is the possibility of interactions between kappa carrageenan double helices and mannan chains. This interacting process is allowed by the flexibility of the mannan chains and structural changes of the kappa carrageenan double helices. The calculations suggest that a disaccharide mannan fragment might be required for recognition. The results of our investigation are in good agreement with a model of gel structure based on experimental data. This approach could be applied to simulate and predict other associations in molecular assemblies.

Purification and characterization of a pig intestinal alpha-limit dextrinase.

Mucosa from the duodenal and jejunal regions of pig small intestine was repeatedly freeze-thaw treated to solubilize an enzyme preparation, enriched in maltase, glucoamylase and alpha-limit dextrinase activities; isomaltase and sucrase remained essentially insoluble during the treatment. Chromatographic procedures, including ion-exchange, gel filtration and hydroxylapatite chromatography of the solubilized preparation, brought to homogeneity an alpha-glucosidase active towards maltose, alpha-limit dextrins and starch in decreasing order, with only a very weak capacity to hydrolyse alpha-1,6-linkages. Michaelis constants and maximal velocities, as well as relative rates of hydrolysis of several substrates, including maltodextrins and alpha-limit dextrins, were determined and served to characterize what seems to be a rather specific alpha-1,4-glucosidase. The participation of this enzyme in the hydrolysis of alpha-limit dextrins and more generally in pathways for starch breakdown in the pig digestive tract is examined and discussed.

Conformational properties of a cycle oligosaccharide: cyclotrikis-(1-->6)-[alphaD-glucopyranosyl-(1-->4)-beta-D-glucopyranosy l].

The title compound is a cyclic oligosaccharide having six glucopyranose residues linked alternatively by alpha-(1-->4) and beta-(1-->6) glycosidic linkages. Like cyclodextrin analogues it is expected to exhibit an internal cavity and to form inclusion complexes with other species. In order to investigate its conformational preferences, an extensive conformational search was carried out using a combination of Metropolis Monte-Carlo (MMC) procedure in the glycosidic torsion angle space and molecular mechanics procedures. To this end a specific program (METROCYCLIX) was developed. To reduce the MMC search, conformational maps of parent disaccharides were considered as starting entries. Fully minimized conformations were gathered into families using a clustering technique based on RMS fitting over the glycosidic torsion angle values. A wide range of local energy minima were identified in spite of ring closure conditions that constrained the structure of the oligosaccharide. Low energy conformers were stabilized by intramolecular interactions between distant residues. From the Bolzmann population of the best structures derived from the clustering results, various average properties were calculated and compared with experimental data obtained by high resolution NMR. Interpretation of these experimental values (heteronuclear coupling constants, rotating frame nuclear Overhauser effects, relaxation times) relies on the use of Karplus like equations (coupling constants) and analysis of the full relaxation rate matrix treatment (ROE). The quality of the molecular modelling strategy used is assessed by the agreement obtained between calculated and measured observables.